Ni2+高效结合肽的筛选与作用研究

利用噬菌体随机十二肽库和金属亲和层析对重金属Ni2 进行结合肽筛选。经4轮生物淘洗、噬菌体扩增和DNA测序,获得一组多肽序列。GenBank Blast分析未发现同源序列,Clustal W多重序列比对也未找到Ni2 金属结合肽结合基序,但可能含有多聚组氨酸(His)2-5。噬菌体单克隆金属离子螯合树脂的亲和力测定和反筛、抑菌解毒试验表明:展示有金属结合肽的噬菌体不仅对Ni2 具有高亲和力,而且对其它金属离子也有作用,Cu2 、Ni2 、Co2 、Zn2 等金属离子对金属结合肽的亲和力显著高于Cd2 和Cr2 ,展示金属结合肽的噬菌体对重金属Ni2 具有一定的耐受和解毒作用。显微形态学观察也显示金属结合肽与金属螯合树脂的作用。对于了解重金属与多肽的相互作用机理以及环境重金属修复等均具有重要意义和价值。     

Ni2+高效结合肽的筛选与作用研究

利用噬菌体随机十二肽库和金属亲和层析对重金属Ni2+进行结合肽筛选。经4轮生物淘洗、噬菌体扩增和DNA测序,获得一组多肽序列。GenBank Blast分析未发现同源序列,Clustal W多重序列比对也未找到Ni2+金属结合肽结合基序,但可能含有多聚组氨酸（His）2－5。噬菌体单克隆金属离子螯合树脂的亲和力测定和反筛、抑菌解毒试验表明:展示有金属结合肽的噬菌体不仅对Ni2+具有高亲和力,而且对其它金属离子也有作用,Cu2+、Ni2+、Co2+、Zn2+等金属离子对金属结合肽的亲和力显著高于Cd2＋和Cr2＋,展示金属结合肽的噬菌体对重金属Ni2+具有一定的耐受和解毒作用。显微形态学观察也显示金属结合肽与金属螯合树脂的作用。对于了解重金属与多肽的相互作用机理以及环境重金属修复等均具有重要意义和价值。     

渭河陕西段沉积物重金属空间分布及来源解析

为了了解渭河陕西段河道沉积物重金属空间分布特征,本研究对渭河陕西段干流及其支流17个采样点沉积物中的10种重金属元素(Cd、Sb、As、Co、Cu、Pb、Ni、Cr、Zn、Mn)含量进行测定及来源辨析。结果表明: 重金属元素Cd、Sb、As、Co、Cu、Pb、Ni、Cr、Zn、Mn的平均含量分别为0.10、1.24、11.73、11.95、24.90、24.91、29.31、54.18、72.74、626.85 mg·kg^-1, 除Cd的变异系数大于1以外,其他元素的变异系数均低于0.5。其中,Cd、Pb、Cr含量于灞河入渭处达到峰值,Co和Mn在黑河入渭处达到峰值,Cu和Zn在清姜河入渭处达到峰值, Sb、As和Ni分别于沙王渡、咸阳铁桥和林家村处达到峰值。相关性分析、主成分分析和聚类分析表明,Cd、Co、Cu、Pb、Ni、Cr、Zn、Mn主要来源于以工业源和生活源为主的污染源;Sb、As主要来源于农业和地球化学污染源。     

临安市雷竹林土壤重金属污染特征及生态风险评价

为了解临安市雷竹林土壤重金属污染特征,采集并测定了160个土壤样品的Hg、As、Cu、Pb、Zn、Cd、Cr、Ni、Co、Mn等重金属含量,采用单因子污染指数和内梅罗综合污染指数对雷竹林土壤重金属污染程度进行分析,并应用Hankanson潜在生态风险指数法对雷竹林土壤重金属潜在生态风险进行评价.结果表明:雷竹林土壤重金属Hg、As、Cu、Pb、Zn、Cd、Cr、Ni、Co、Mn的平均含量分别为0.16、7.41、34.36、87.98、103.98、0.26、59.12、29.56、11.44、350.26mg·kg-1,Pb、Cd、Zn和Cu平均值超过浙江省土壤背景值,分别是对应背景值的2.89、1.70、1.12、1.12倍.经单因子污染指数评价,不同重金属元素的平均污染程度大小依次为Pb＞Cd＞Cu=Zn＞Hg＞As＞Ni＞Co＞Cr＞Mn,其中Pb有中度污染,Cd、Cu和Zn有轻度污染.经内梅罗综合污染指数评价,160个样点都受到不同程度的重金属污染,轻度污染、中度污染和重度污染水平所占比率分别为55.6％、29.4％和15.0％.各重金属单因子潜在生态风险指数平均值评价结果显示,只有Cd污染达到中等生态风险,其他重金属均为轻微生态风险,而局部采样点Cd和Hg单因子潜在生态风险指数最大值分别达到256.82和187.33,存在很强生态风险.重金属综合因子潜在生态风险指数评价结果表明,临安市雷竹林土壤整体上存在轻微生态风险.     

公路绿化植物油松(Pinus tabulaeformis)和小叶杨(Populus simonii)对重金属元素的吸收与积累

对内蒙古西部公路绿化植物油松（Pinus tabulaeformis）、小叶杨（Populus simonii）及其根际土壤中重金属元素（Cd、Hg、Pb、Cu、Zn、Ni、Cr）和类金属元素（As和Se）含量以及根际土壤重金属（Cu、Zn、Pb、Ni和Cr）形态、土壤pH值进行了测定。对比分析了公路沿线不同绿化植物及其不同器官对重金属元素的吸收与积累特征。结果表明：绿化植物根际土壤对重金属元素的吸附及污染程度以Cd为最高。随原子序数的递增,小叶杨和油松两种植物的根部和茎叶两种营养器官中重金属的含量均表现出“N”字形变动趋势。而且重金属元素在不同植物不同器官中的含量具有Zn〉Cu〉Ni,Cr,As,Pb〉Cd〉Hg的基本规律。小叶杨茎叶对重金属元素Cr、Ni和Pb的富集能力较根部为强,油松茎叶对重金属元素Cr、Ni、Cu和Pb的富集能力较根部为强。绿化植物根际土壤重金属元素有效态占总量百分比的大小序列为Zn〉Pb〉Ni、Cr〉Cu,与重金属元素在不同植物不同器官中的含量大小序列Zn〉Cu〉Ni、Cr、As、Pb〉Cd〉Hg并非趋于一致。公路绿化植物对根际土壤中重金属元素的吸收和积累与重金属元素有效态所占的比例有关。     

东苕溪沉积物重金属生态风险评价和源解析

由于重金属毒性大, 且易在食物链中富集, 沉积物中的重金属会对水体生态环境造成严重污染, 因此对东苕溪23个采样点表层沉积物中主要的重金属As、Cd、Co、Cr、Cu、Pb、Zn、Mn和Ni含量及分布特征进行研究, 分析各种重金属来源, 并对重金属污染状况进行生态风险评价。结果表明: 东苕溪沉积物重金属平均浓度Mn>Pb> Zn> Cr> As> Cu> Ni> Co> Cd, As、Cd、Co、Cu、Pb、Zn和Mn的平均浓度均高于它们的环境背景值。多元统计分析结果表明沉积物中Cd、Co、Cr、Mn和Ni可能来源于自然环境, Cu、Pb 和Zn可能来源于生活污水和工业废水的排放, Pb还源于交通工具尾气和柴油机械排放的废弃物, As主要来源于农业活动, 例如化肥和农药的使用。地积累指数法和潜在生态危害评价法的结果表明, 东苕溪沉积物中的重金属整体上呈现中等的潜在生态危害, 东苕溪沉积物中的主要污染物为As和Cd, 由于东苕溪流域散布着大量的农田和一些工业园区, 农田中使用的肥料农药和工业活动中排放的废弃物是造成As和Cd含量高的原因。     

海口城市土壤重金属污染特征与生态风险评估

对海口城市土壤重金属含量、空间分布特征与赋存形态进行了研究,并评估了其生态风险。结果表明,海口城市土壤重金属Hg、As、Cd、Cu、Cr、Ni、Pb、Zn的平均含量分别为0.073、3.82、0.25、26.7、92.4、52.5、29.1和84.1 mg·kg-1。与海口土壤背景值相比,海口城市土壤明显富集重金属Hg、As、Cd、Cu、Cr、Ni、Pb和Zn,受到一定程度重金属污染。Cr、Ni、Cu、Cd和Zn元素主要在郊区富集,Pb主要在路边绿化带中富集,Hg主要在生活区富集。海口城市土壤中Zn、As、Cr、Cu和Ni以残渣态为主,Hg主要以强有机态和残渣态存在,Pb主要以铁锰氧化态和残渣态存在,而Cd则主要以生物可利用态为主。生态风险评价结果显示,海口城市土壤重金属综合生态风险属于微弱水平,但Cd和Hg污染应引起重视。     

Cd2+结合肽的筛选及与重金属离子的亲和作用*

利用噬菌体随机十二肽库和亲和层析技术对重金属Cd进行亲和筛选,共获得两条Cd结合肽序列。将展示有Cd²⁺结合肽的噬菌体单克隆扩增物对不同重金属离子(Cd²⁺、Cr²⁺、Cu²⁺、Co²⁺、Zn²⁺、Ni²⁺)螯合的树脂进行亲和测定,结果表明Cu²⁺、Co²⁺、Zn²⁺、Ni^2+<     

刺楸年轮中重金属含量动态变化及富集特性

为探索刺楸对受污染土壤重金属的富集和修复效应, 以南京栖霞山的乡土树种刺楸及其根际周边土壤为研究对象, 截取其根基部年轮盘及根际土壤样本, 采用ICP-AES法测定年轮及土壤样本中重金属(Cu、Cd、Cr、Mn、Ni、Pb、Zn)元素含量。结果表明: 栖霞山样地中的土壤受Mn、Pb和Zn污染最为严重, 存在Cu、Cd、Mn、Pb、Zn元素的高度复合污染, Cd、Cr、Cu、Ni、Zn在土壤和年轮中存在相关性, Mn和Pb则没有表现出明显的相关性; 刺楸修复受Cd、Mn、Pb、Zn污染的土壤效果并不显著, 更适用于Cr、Cu、Ni污染的土壤修复; 鉴于Cu元素含量变化特征, 刺楸也可以作为反映当地污染历史的记录载体; 刺楸年轮中的重金属元素之间存在交互作用, 其中Cd与Zn元素含量高度相关(r=0.984, p<0.01), 在刺楸年轮吸收重金属元素的过程中, Cu与Cd、Cr、Mn、Zn元素具有协同作用, Mn元素对其他元素有一定的拮抗作用。     

贵阳花溪区石灰土林地土壤重金属含量特征及其污染评价

选取贵阳市花溪区典型石灰土林地土壤作为研究对象,分析了林地石灰岩和土壤中7种重金属(Cu、Zn、Mn、Cd、Ni、Pb、Co)的含量特征,以贵州省土壤背景值和全国石灰(岩)土类背景值为评价标准进行林地土壤重金属污染评价和潜在生态风险评价.结果表明:林地石灰岩以Pb的平均含量(40.21mg·kg-1)最高,Zn的(5.78 mg·kg-1)最低,7种重金属平均含量高低顺序为:Pb＞Ni＞Mn＞Co＞Cu＞Cd＞Zn;林地土壤中以Mn的平均含量(451.16 mg·kg-1以上)最高,Cd的(2.87mg·kg-1以下)最低,7种重金属含量的变异系数在8.57%～63.10%之间,Zn的平均含量明显低于贵州省土壤背景值和全国石灰(岩)土类背景值,Cu、Mn、Cd、Pb、Ni、Co的平均含量高于或接近于贵州省土壤背景值和全国石灰(岩)土类背景值.Cu、Zn、Mn、Ni、Co,Ni与Pb,Cd与Pb,Cd与Co来源相同的可能性较大,而Cd与Cu、Ni,Pb与Mn、Cu、Co的来源不同;石灰土偏碱性,富含Ca、Mg元素,有利于重金属Cd、Pb的累积.单因子污染指数和多因子综合指数(内梅罗指数法)与Hakanson潜在生态危害指数的评价结果一致,林地土壤重金属综合污染指数在3.67以上,达到重污染程度,以Cd的污染指数(4.94以上)最高,污染程度最为严重,其次是Pb(1.82以上),Zn、Mn污染程度最低,林地土壤重金属潜在生态危害指数(RI)为173.75以上,为中度生态危害程度,产生最大生态危害的是Cd,其次是Pb、Ni、Co、Cu,危害程度最小的是Mn、Zn,在相同的成土母岩和人为活动影响下,无林地土壤重金属的综合污染指数和潜在生态危害指数均明显高于有林地.     

Selection of staphylococcal enterotoxin B (SEB)-binding peptide using phage display technology

In this study, peptides were selected to recognize staphylococcal enterotoxin B (SEB) which cause food intoxication and can be used as a biological war agent. By using commercial M13 phage library, single plaque isolation of 38 phages was done and binding affinities were investigated with phage-ELISA. The specificities of the selected phage clones showing high affinity to SEB were checked by using different protein molecules which can be found in food samples. Furthermore, the affinities of three selected phage clones were determined by using surface plasmon resonance (SPR) sensors. Sequence analysis was realized for three peptides showing high binding affinity to SEB and WWRPLTPESPPA, MNLHDYHRLFWY, and QHPQINQTLYRM amino acid sequences were obtained. The peptide sequence with highest affinity to SEB was synthesized with solid phase peptide synthesis technique and thermodynamic constants of the peptide-SEB interaction were determined by using isothermal titration calorimetry (ITC) and compared with those of antibody-SEB interaction. The binding constant of the peptide was determined as 4.2 ± 0.7 × 10⁵ M⁻¹ which indicates a strong binding close to that of antibody.     

Enhanced bioaccumulation of heavy metal ions by bacterial cells due to surface display of short metal binding peptides

Metal binding peptides of sequences Gly-His-His-Pro-His-Gly (named HP) and Gly-Cys-Gly-Cys-Pro-Cys-Gly-Cys-Gly (named CP) were genetically engineered into LamB protein and expressed in Escherichia coli. The Cd2+-to-HP and Cd2+-to-CP stoichiometries of peptides were 1:1 and 3:1, respectively. Hybrid LamB proteins were found to be properly folded in the outer membrane of E. coli. Isolated cell envelopes of E. coli bearing newly added metal binding peptides showed an up to 1.8-fold increase in Cd2+ binding capacity. The bioaccumulation of Cd2+, Cu2+, and Zn2+ by E. coli was evaluated. Surface display of CP multiplied the ability of E. coli to bind Cd2+ from growth medium fourfold. Display of HP peptide did not contribute to an increase in the accumulation of Cu2+ and Zn2+. However, Cu2+ ceased contribution of HP for Cd2+ accumulation, probably due to the strong binding of Cu2+ to HP. Thus, considering the cooperation of cell structures with inserted peptides, the relative affinities of metal binding peptide and, for example, the cell wall to metal ion should be taken into account in the rational design of peptide sequences possessing specificity for a particular metal.     

Linear free-energy analysis of mercury(II) and cadmium(II) binding to three-stranded coiled coils

Investigators have studied how proteins enforce nonstandard geometries on metal centers to assess the question of how protein structures can define the coordination geometry and binding affinity of an active-site metal cofactor. We have shown that cysteine-substituted versions of the TRI peptide series [AcG-(LKALEEK)(4)G-NH(2)] bind Hg(II) and Cd(II) in geometries that are different from what is normally found with thiol ligands in aqueous solution. A fundamental question has been whether this structural perturbation is due to protein influence or a change in the metal geometry preference. To address this question, we have completed linear free-energy analyses that correlate the association of three-stranded coiled coils in the absence of a metal with the binding affinity of the peptides to the heavy metals, Hg(II) and Cd(II). In this paper, six new members of this family have been synthesized, replacing core leucine residues with smaller and less hydrophobic residues, consequently leading to varying degrees of self-association affinities. At the same time, studies with some smaller and longer sequenced peptides have also been examined. All of these peptides are seen to sequester Hg(II) and Cd(II) in an uncommon trigonal environment. For both metals, the binding is strong with micromolar dissociation constants. For binding of Hg(II) to the peptides, the dissociation constants range from 2.4 x 10(-)(5) M for Baby L12C to 2.5 x 10(-)(9) M for Grand L9C for binding of the third thiolate to a linear Hg(II)(pep)(2) species. The binding of Hg(II) to the peptide Grand L9C is similar in energetics for metal binding in the metalloregulatory protein, mercury responsive (merR), displaying approximately 50% trigonal Hg(II) formation at nanomolar metal concentrations. Approximately, 11 kcal/mol of the Hg(II)(Grand L9C)(3)(-) stability is due to peptide interactions, whereas only 1-4 kcal/mol stabilization results from Hg(II)(RS)(2) binding the third thiolate ligand. This further validates the hypothesis that the favorable tertiary interactions in protein systems such as merR go a long way in stabilizing nonnatural coordination environments in biological systems. Similarly, for the binding of Cd(II) to the TRI family, the dissociation constants range from 1.3 x 10(-)(6) M for Baby L9C to 8.3 x 10(-)(9) M for TRI L9C, showing a similar nature of stable aggregate formation.     

An allosteric mechanism controls antigen presentation by the H-2K(b) complex.

The mechanism of assembly/dissociation of a recombinant water-soluble class I major histocompatibility complex (MHC) H-2Kb molecule was studied by a real-time fluorescence resonance energy transfer method. Like the H-2Kd ternary complex [Gakamsky et al. (1996) Biochemistry 35, 14841-14848], the interactions among the heavy chain, beta2-microglobulin (beta2m), and antigenic peptides were found to be controlled by an allosteric mechanism. Association of the heavy chain with beta2m increased peptide binding rate constants by more than 2 orders of magnitude and enhanced affinity of the heavy-chain molecule for peptides. Interaction of peptides with the heavy-chain binding site, in turn, increased markedly the affinity of the heavy chain for beta2m. Binding of peptide variants of the ovalbumin sequence (257-264) to the heavy chain/beta2m heterodimer was found to be a biphasic reaction. The fast phase was a second-order process with nearly the same rate constants as those of binding of peptides derived from the influenza virus nucleoprotein 147-155 to the H-2Kd heavy chain/beta2m heterodimer [(3.0 +/- 1.0) x 10(-6) M-1 s-1 at 37 degrees C]. The slow phase was a result of both the ternary complex assembly from the "free" heavy chain, beta2m, and peptide as well as an intramolecular conformational transition within the heavy chain/beta2m heterodimer to a peptide binding conformation. Biexponential kinetics of peptide or beta2m dissociation from the ternary complex were observed. They suggest that it can exist in two conformations. The rate constants of beta2m dissociation from the H-2Kb ternary complex were, in the limits of experimental accuracy, independent of the structure of the bound peptide, though their affinities differed by an order of magnitude. Dissociation of peptides from the Kb heavy chain was always faster than from the ternary complexes, yet the heavy chain/peptide complexes were considerably more stable compared with their Kd/nucleoprotein peptide counterparts.     

Novel p16 binding peptide development for p16‐overexpressing cancer cell detection using phage display

Protein p^16INK4a (p16) is a well‐known biomarker for diagnosis of human papillomavirus (HPV) related cancers. In this work, we identify novel p16 binding peptides by using phage display selection method. A random heptamer phage display library was screened on purified recombinant p16 protein‐coated plates to elute only the bound phages from p16 surfaces. Binding affinity of the bound phages was compared with each other by enzyme‐linked immunosorbent assay (ELISA), fluorescence imaging technique, and bioinformatic computations. Binding specificity and binding selectivity of the best candidate phage‐displayed p16 binding peptide were evaluated by peptide blocking experiment in competition with p16 monoclonal antibody and fluorescence imaging technique, respectively. Five candidate phage‐displayed peptides were isolated from the phage display selection method. All candidate p16 binding phages show better binding affinity than wild‐type phage in ELISA test, but only three of them can discriminate p16‐overexpressing cancer cell, CaSki, from normal uterine fibroblast cell, HUF, with relative fluorescence intensities from 2.6 to 4.2‐fold greater than those of wild‐type phage. Bioinformatic results indicate that peptide ‘Ser‐His‐Ser‐Leu‐Leu‐Ser‐Ser’ binds to p16 molecule with the best binding score and does not interfere with the common protein functions of p16. Peptide blocking experiment shows that the phage‐displayed peptide ‘Ser‐His‐Ser‐Leu‐Leu‐Ser‐Ser’ can conceal p16 from monoclonal antibody interaction. This phage clone also selectively interacts with the p16 positive cell lines, and thus, it can be applied for p16‐overexpressing cell detection. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Identification of anti-TNFalpha peptides with consensus sequence

Phage displayed peptide library was used to select tumor necrosis factor alpha (TNFalpha) binding peptides. After three sequential rounds of biopanning, some linear TNFalpha-binding peptides were identified from a 12-mer peptide library. A consensus sequence (L/M)HEL(Y/F)(L/M)X(W/Y/F), where X might be variable residue, was deduced from sequences of these peptides. The phages bearing these peptides showed specific binding to immobilized TNFalpha, with over 80% of phages bound being competitively eluted by free TNFalpha. To confirm the binding activity and to explore further functional properties, three peptides with typical structure were selected and expressed as GST-fused protein. These recombinant peptides effectively competed for [125I]TNFalpha binding to TNFR1 in a dose-dependent manner, with IC(50) from 10 to 160 microM. Furthermore, the GST-fused derivatives showed inhibitory effects on TNFalpha-induced cytotoxicity. Taken together, these data demonstrate that the TNFalpha-binding peptides are effective antagonists of TNFalpha and the deduced motif might be useful in development of novel low molecular weight anti-TNFalpha drugs.     

Intramolecular azo-bridge as a cystine disulfide bond surrogate: Somatostatin-14 and brain natriuretic peptide (BNP) analogs

Cystine disulfide bond is a common feature in numerous biologically active peptides and proteins and accordingly its replacement by various surrogates presents a potential route to obtain analogs with improved pharmacokinetic characteristics. The purpose of the present study was to assess whether an azo-bridge can serve as such a surrogate. In view of the marked clinical significance of somatostatin and the brain natriuretic peptide (BNP) we choose these peptides as a model. Three cyclic-azo somatostatin analogs and three cyclic-azo BNP analogs were effectively prepared in solution through azo bond formation between p-amino phenylalanine and His or Tyr residues that were positioned in the peptide sequences in place of the native Cys residues. The peptides binding affinities to the sst? and ANP-receptor (NPR-A) expressed on rat acinar pancreating carcinoma AR4-2J cell membranes and HeLa cells, respectively, were examined. The somatostatin analogs displayed good to moderate affinities to the rat sst? in the nM range with best results obtained with peptide 2, that is, IC?? = 8.1 nM. Molecular dynamics simulations on these peptides suggests on a correlation between the observed binding potencies and the degree of conformational space overlapping with that of somatostatin. The BNP analogs exhibited binding affinities to the NPR-A in the nM range with best results obtained with BNP-1, that is, IC?? = 60 nM.     

Detection of weakly interacting anti-carbohydrate scFv phages using surface plasmon resonance

Methods to characterise and confirm specificity of scFv displayed on phages are important during panning procedures, especially when selecting for antibody fragments with weak affinities in the millimole to micromole range. In this report the surface plasmon resonance (SPR) biosensor was used to study and verify specificity of phages displaying weak anti-carbohydrate scFvs. The variable immunoglobulin light (VL) and heavy (VH) chain genes of the weak monoclonal antibody 39.5 were amplified and cloned into a phagemid and displayed as a scFv-pIII fusion protein on filamentous phage. This monoclonal antibody recognises with weak affinity the structural sequence Glcalpha1-4Glc present in a variety of carbohydrate molecules. Injection of the 39.5 phages over a biosensor chip immobilised with a (Glc)4-BSA conjugate confirmed selective binding of the scFv to its antigen. Inhibition studies verified the specificity. These results clearly show that SPR technology can be used to evaluate in terms of binding and specificity weakly interacting scFv displayed on the phage surface.     

Affinity for the cognate monoclonal antibody of synthetic peptides derived from selection by phage display. Role of sequences flanking thebinding motif.

Randomized peptide sequences displayed at the surface of filamentous phages are often used to select antibody ligands. The selected sequences are generally further used in the form of synthetic peptides; however, as such, their affinity for the selecting antibody is extremely variable and factors influencing this affinity have not been fully deciphered. We have used an f88.4 phage-displayed peptide library to identify ligands of mAb 11E12, an antibody reactive to human cardiac troponin I. A majority of the sequences thus selected showed a (T/A/I/L) EP(K/R/H) motif, homologous to the Y-TEPH motif identified by multiple peptide synthesis as the critical motif recognized by mAb 11E12 in the peptide epitope. A set of 15-mer synthetic peptides derived from the phage-selected sequences was used in BIACORE to characterize their interaction with mAb 11E12. Most peptides exhibited affinities in the 7-26 nM range. These affinities represented, however, only 1.9-7. 5% of the affinity of the 15-mer peptide epitope. In circular dichroism experiments, the peptide epitope showed a propensity to have some stabilized conformation, whereas a low-affinity peptide selected by phage-display did not. To try to decipher the molecular basis of this difference in affinity, new peptides were prepared by grafting the N- or the C-terminal sequence of the peptide epitope to the Y-TEPK motif of a low-affinity peptide selected by phage-display. These hybrid peptides showed marked increases both in affinity (as assessed using BIACORE) and in inhibitory potency (as assessed in competition ELISA), compared with the parent sequence. Thus, the sequences flanking the motif, although not containing critical residues, convey some determinants necessary for high affinity. The affinity of a given peptide strongly depends on its capacity to maintain the antigenically reactive structure it has on the phage, implying that it is impossible to predict whether high- or low-affinity peptides will be obtained from phage display.